Robust production of recombinant phosphoproteins using cell-free protein synthesis

Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure–function relationships, kinase signalling networks and kinase inhibitor drugs.


Supplementary Tables
: Identification of N-terminal peptides from sfGFPs reporting incorporation of amino acids at positions S2 or E17 of sfGFP-S2TAG and sfGFP-E17TAG. All peptides are reported with a FDR cut-off of 1% and a minimum delta score of 5. Amino acids incorporated at TAG are in red. Peptides reporting skipping at TAG are in bold. Peptides reporting incorporation of K or R are marked with (K/R)* indicating that the amino acid K or R was cleaved off during tryptic digestion. Abbreviations: (de) deamidation, (ox) oxidation, (ph) phosphorylation.    Bacterial cell pellets were stored at -80°C until extract preparation.

CFPS extract preparation
Cell pellets were thawed on ice and resuspended in ice cold S30 buffer using a cell weight to buffer ratio of 1.0g wet cell mass to 0.8ml S30 buffer. Cells were lysed on ice by sonication with a model Q125 sonicator (QSonica LLC, Newtown, CT). The lysis program consisted of three 45s bursts performed at 50% amplitude and 59s pause between subsequent bursts. The total deposited energy of approximately 900 Joules 3 . Lysate was supplemented with an additional 2mM Dithiothreitol (DTT) and cell debris was removed with a 10min spin performed at 12,000 x g at 4°C. The clarified cell extract was incubated for 1h at 37°C while shaking at 220 RPM and insoluble material was removed by centrifugation performed at 12,000 x g for 30min at 4°C. The supernatant was flash-frozen in liquid nitrogen and stored at -80°C until use for CFPS.

Protein quantification of sfGFP and MEK1
Expression of active sfGFP proteins in CFPS extracts was quantified by fluorescence spectroscopy. Briefly, CFPS reactions were diluted 50x with 50mM HEPES buffer pH 7.

Protein digestion for mass spectrometry
Digestion of sfGFPs: Affinity purified and buffer exchanged protein was digested with trypsin according to an optimized protocol for in solution digestion of sfGFP reported by Aerni 8

Label-free quantitation of amino acid incorporation
Label-free quantitation of amino acid incorporation for sfGFP was performed by spectra counting (Supplementary Fig. 2, Supplementary Table 2) and using MS1 intensity based quantitation (Supplementary Table 3). The MS1 peak intensities for each charge state of all peptides reported in Supplementary Table 1 were determined with Skyline software 9 v.
2.5.0.6157 and the sum of all precursor intensities for each peptide was exported into Microsoft Excel 2013 for further processing. The peptide FEGDTLVNR corresponding to amino acid residue 114-122 of the sfGFP reporter was included to normalize ion intensities between GFP samples as reported earlier 2 . To quantitate the incorporation of amino acids at position S2 and E17, it was assumed that the sum of all normalized ion intensities reporting incorporation of amino acids at position S2 or E17 adds up to 100% and that the ionization efficiency for each peptide is the same. Note that peptides reporting incorporation of K in the E17=TAG sfGFP were excluded from the analysis because cleavage with trypsin (cleaving C terminal of K and R) generates shorter peptides with significantly different ionization properties. The incorporation of each detected amino acid was then calculated as a percentage relative to the sum of the total ion intensities of all peptides for S2 sfGFP and for all peptides except peptides reporting incorporation of K at position E17=TAG (Supplementary Table 3, Supplementary Figs. 3 and 4).